IDENTIFICATION OF INTERACTIONS BETWEEN NITRIC OXIDE SYNTHASE AND CALMODULIN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。一氧化氮合酶（NOS）在从平滑肌松弛到神经元中的细胞-细胞信号传导的多种生理功能中起作用。这种酶催化精氨酸氧化产生多功能小分子一氧化氮（NO）。NO的不受调节的产生可导致许多病理状况，如中风和阿尔茨海默病。有三种不同的NOS亚型，它们受钙调素（CaM）的差异调节。在神经元和内皮细胞中发现的异构体由Ca 2 +/CaM的可逆结合调节，而在巨噬细胞中发现的异构体由Ca 2 +/CaM的可逆结合调节。（诱导型）与CaM结合紧密到似乎是不可逆的。1先前的研究已经证明，神经元型和诱导型NOS具有非常不同的要求，对于这些要求，CaM的Ca 2+结合位点必须与Ca 2+结合以实现激活，并表明钙调素和这两种NOS亚型之间的相互作用是在相反的方向。2，3代替这些蛋白质的分子结构，理解这种现象需要研究，在逐步解开的相互作用，然后拼凑信息在一起。Stevens-Truss实验室的长期目标是了解CaM与NOS结合和调节的机制。质谱法提供了一种可以用来解开这些相互作用的工具。传统的蛋白质指纹图谱的方法，加上质谱法计划在这个项目中使用，试图开发方法来研究钙调素和各种NOS亚型之间的相互作用。这项研究很重要，因为（i）它将增加我们对NOS整体折叠结构的理解，因为它与CaM结合和激活有关，（ii）它将揭示CaM在结合和激活30多种不同蛋白质中仍然神秘的作用，（iii）它将推动设计NOS亚型选择性抑制剂的努力。此外，这些研究将有助于我们理解破坏蛋白质-蛋白质相互作用的因素，如光能碰撞。一氧化氮合酶（NOS）在从平滑肌松弛到神经元中的细胞-细胞信号传导的多种生理功能中起作用。这种酶催化精氨酸氧化产生多功能小分子一氧化氮（NO）。NO的不受调节的产生可导致许多病理状况，如中风和阿尔茨海默病。有三种不同的NOS亚型，它们受钙调素（CaM）的差异调节。在神经元和内皮细胞中发现的异构体由Ca 2 +/CaM的可逆结合调节，而在巨噬细胞中发现的异构体由Ca 2 +/CaM的可逆结合调节。（诱导型）与CaM结合紧密到似乎是不可逆的。1先前的研究已经证明，神经元型和诱导型NOS具有非常不同的要求，对于这些要求，CaM的Ca 2+结合位点必须与Ca 2+结合以实现激活，并表明钙调素和这两种NOS亚型之间的相互作用是在相反的方向。2，3代替这些蛋白质的分子结构，理解这种现象需要研究，在逐步解开的相互作用，然后拼凑信息在一起。Stevens-Truss实验室的长期目标是了解CaM与NOS结合和调节的机制。质谱法提供了一种可以用来解开这些相互作用的工具。传统的蛋白质指纹图谱的方法，加上质谱法计划在这个项目中使用，试图开发方法来研究钙调素和各种NOS亚型之间的相互作用。这项研究很重要，因为（i）它将增加我们对NOS整体折叠结构的理解，因为它与CaM结合和激活有关，（ii）它将揭示CaM在结合和激活30多种不同蛋白质中仍然神秘的作用，（iii）它将推动设计NOS亚型选择性抑制剂的努力。此外，这些研究将有助于我们理解破坏蛋白质-蛋白质相互作用的因素，如光能碰撞。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitric oxide synthase (NOS) plays a role in a variety of physiological functions from smooth muscle relaxation to cell-cell signaling in neurons. This enzyme catalyzes the oxidation of arginine to generate the multifunctional small molecule nitric oxide (¿NO). The unregulated production of ¿NO can lead to a number of pathological conditions such as stroke and Alzheimer¿s disease. There are three distinct isoforms of NOS that are differentially regulated by calmodulin (CaM). The isoforms found in neuronal and endothelial cells are regulated by the reversible binding of Ca2+/CaM, while the form found in macrophages (inducible) binds CaM tight enough to be seemingly irreversible.1 Previous research has demonstrated that the neuronal and the inducible NOS have very different requirements for which Ca2+ binding site of CaM must be Ca2+ bound in order to achieve activation, and suggests that the interactions between CaM and these two NOS isoforms is in opposite orientation.2,3 In lieu of molecular structures of these proteins, understanding this phenomenon requires studies geared at the gradual unraveling of the interactions and then piecing the information back together. The long-term objective of Dr. Stevens-Truss¿ laboratory is to understand the mechanisms underlying CaM¿s binding to and regulation of NOS. Mass spectrometry offers a tool that can be used to unravel these interactions. Traditional protein fingerprinting methodologies coupled with mass spectrometry are planned for use in this project, in an attempt to develop ways to study the interactions between CaM and the various NOS isoforms. This research is important because (i) it will increase our understanding of the overall folded structure of NOS as it relates to CaM binding and activation, (ii) it will shed light on the still enigmatic role of CaM in binding and activating over 30 different proteins, and (iii) it will advance efforts to design NOS isoform selective inhibitors. Moreover, these studies will aid in our understanding of factors that disrupt protein-protein interactions such as light energy collisions. Nitric oxide synthase (NOS) plays a role in a variety of physiological functions from smooth muscle relaxation to cell-cell signaling in neurons. This enzyme catalyzes the oxidation of arginine to generate the multifunctional small molecule nitric oxide (¿NO). The unregulated production of ¿NO can lead to a number of pathological conditions such as stroke and Alzheimer¿s disease. There are three distinct isoforms of NOS that are differentially regulated by calmodulin (CaM). The isoforms found in neuronal and endothelial cells are regulated by the reversible binding of Ca2+/CaM, while the form found in macrophages (inducible) binds CaM tight enough to be seemingly irreversible.1 Previous research has demonstrated that the neuronal and the inducible NOS have very different requirements for which Ca2+ binding site of CaM must be Ca2+ bound in order to achieve activation, and suggests that the interactions between CaM and these two NOS isoforms is in opposite orientation.2,3 In lieu of molecular structures of these proteins, understanding this phenomenon requires studies geared at the gradual unraveling of the interactions and then piecing the information back together. The long-term objective of Dr. Stevens-Truss¿ laboratory is to understand the mechanisms underlying CaM¿s binding to and regulation of NOS. Mass spectrometry offers a tool that can be used to unravel these interactions. Traditional protein fingerprinting methodologies coupled with mass spectrometry are planned for use in this project, in an attempt to develop ways to study the interactions between CaM and the various NOS isoforms. This research is important because (i) it will increase our understanding of the overall folded structure of NOS as it relates to CaM binding and activation, (ii) it will shed light on the still enigmatic role of CaM in binding and activating over 30 different proteins, and (iii) it will advance efforts to design NOS isoform selective inhibitors. Moreover, these studies will aid in our understanding of factors that disrupt protein-protein interactions such as light energy collisions.